Project Summary/Abstract Spatial control of membrane traffic is essential for the morphogenesis and maintenance of polarized cell types such as epithelia and neurons, and their physiological functions in tissue homeostasis and neurotransmission. Loss of cell polarity and defects in membrane traffic underlie the pathogenesis of many diseases including cancer, neurodegeneration and metabolic disorders. The RATIONALE for our studies is that a mechanistic knowledge of the spatial organization and regulation of membrane traffic is critical for the design of new treatments and regenerative therapies. Many advances have been made in understanding membrane traffic at points of origin (protein sorting, vesicle formation) and destination (vesicle docking/fusion). However, KEY CHALLENGES remain in understanding how long-range transport is spatially controlled en route to destination. Our CENTRAL HYPOTHESIS is that septins, a unique family of GTP-binding proteins that associate with distinct subsets of microtubules (MTs) and membrane domains, comprise a novel regulatory module for the spatial guidance of membrane traffic. Recent findings from our laboratory show that septins modulate motor (kinesins, dynein) interactions with MTs and membranes. Driven by these advances, we will address KEY QUESTIONS about septin functions and the spatial control of membrane traffic in epithelia and neurons. We will investigate whether there is specificity between septins and distinct routes of polarized traffic (e.g., apical vs. basolateral). We will distinguish between septin roles in the spatial orientation of MTs and the modulation of motor motility on MT tracks, which will be mechanistically studied with structure-function approaches. Based on new evidence of septin association with endolysosomes, we will investigate how lysosome trafficking and positioning is regulated by septins. We will test the hypothesis that endolysosomal septins scaffold the recruitment and activation of dynein-dynactin, and examine whether septins are involved in the coordination of kinesin and dynein motors. Lastly, we will investigate how septin association with MTs or membranes is controlled, focusing on phosphorylation of septins by signaling kinases as a switch mechanism between MT- and membrane-associated functions. We will pursue these projects using innovative tools and cutting-edge approaches including in vitro reconstitution, single-molecule and super-resolution microscopy, and 3D organoid culture. Outcomes will provide new insights into the mechanisms that direct the transport of membrane vesicles and endolysosomes in response to morphogenetic and metabolic signals. The proposed studies will also advance our understanding of septins as spatial regulators of intracellular organization, bearing significance on diseases triggered and/or exacerbated by abnormalities in septin expression.